The central hypothesis driving Project 1 is that the clinical efficacy of magnetic nanoparticle (mNP)-based cancer therapies can be substantially enhanced by precise targeting ofthe particles to cancer cells. Previous efforts using antibody and antibody fragments to target nanoparticles has yielded mixed results, and the fragmented nature ofthe related literature does not permit drawing informative conclusions that might guide future studies. To remedy this stark deficit of knowledge regarding targeting of mNP cancer therapies. Project 1 will, in a controlled fashion, conjugate genetically engineered single-chain antibody fragments (scFvs) to the surface of custom-designed mNPs. These mNP immunoconjugates will be subjected to rigorous physical, biochemical, and functional characterization. Upon establishing stringent control of mNP immunoconjugate manufacturing and characterization processes, controlled studies will systematically evaluate three key parameters believed to be critical for clinical efficacy of targeted mNP cancer therapies: (1) mNP specificity and affinity for target cells; (2) the subcellular localization of mNP upon reaching their targets; and (3) the relationship between mNP size and cellular/tumor accumulation of mNP, as assessed in two different cancer modelsbreast cancer and ovarian cancer. Aim 1 will test the hypothesis that simultaneously targeting two different cancer-associated cell surface receptors with a single mNP will increase avid interactions between targeted particles and cancer cells, thereby improving specificity and biodistribution. Aim 2 will test the hypothesis that mNPs are targeted to cancer-antigen-expressing cells more effectively by internalizing antibodies than by non-internalizing antibodies. Aim 3 will test the hypothesis that smaller (10-20 nm) mNP immunoconjugates accumulate in antigen-expressing tumors to a higher level than larger (50-100 nm) mNP immunoconjugates. The objectives of these aims will be achieved by integrating knowledge of antibody-engineering technologies, experience with bioconjugation chemistry, and expertise with in vitro analysis of cellular mNP accumulation. The project will draw on the Nanoparticle Development, Production, and Characterization Core for mNP fabrication, on Projects 3 & 4 for coordinated testing of mNP immunoconjugates in vivo, and on the Toxicology, Pathology, and Biodistribution Core and the Biostatistics, Data Analysis, and Computation Core for analysis of those in vivo results.